HPV is the major etiologic factor in cervical cancer and is found in the majority of cervical tumors. Although infection with oncogenic forms of HPV are common among young women, less than 1% of those infected develop cervical cancer.1, 2, 3 Both the length of the infection and a high viral load several years before diagnosis have been shown to increase the risk of developing cervical cancer.4, 5, 6
HPV has a number of means by which it is able to reduce the immune response of the host or evade the immune system. For instance, since HPV does not infect antigen presenting cells (APCs) of the epithelium or lyse keratinocytes, it reduces the opportunity for the APC to present virally derived antigens to the immune system. During the infection, HPV never reaches the blood vessels, further limiting the exposure to the APCs. At the very late stages of invasive cancer, when tumor cells have penetrated the basal cell membrane and could present a suitable immune target, the main fraction of the viral genomes are integrated in the host genome and only a small fraction remains in episomal form. Viral gene expression also appears to have evolved as to minimize exposure of viral proteins to the immune system, with only a small number of the proteins being expressed in the early phases of propagation. It has even been proposed that the codon usage of HPV has evolved in such a way that the growth of viral particles in mammalian cells would be rate limited by the low availability of certain tRNAs.7 Interferons (IFN) α and β are usually induced by viral infections, but the oncoproteins E6 and E7 have the ability to inhibit this induction.8, 9 Further, by preventing APCs like dendritic cells (DC) to mature, E7 generates a tolerogenic rather than immunogenic reaction.10, 11, 12 As HPV 16 E7 can persist for a number of years in the infected cells, there is ample time for the process of tolerization to occur. Thus, by persisting in a manner similar to a self-antigen, E7 manages to escape a positive adaptive immune response. At the structural level, E7 shows similarity to several human proteins and such sharing of motifs may also reduce immunogenicity.13 Both E6 and E7 have the ability to reduce the immunostimulatory IFN-γ production in the NK cells.14 Additional means for immune evasion of HPV include dysregulation of both the MHC class I antigen15 and the cytokine signals in the tumors.16
Despite the multifaceted ability of HPV to evade or alter the immune system of the host, data from immunocompromised patients17 show that the immune system may affect the outcome of an infection and thereby the development of cervical tumors. Differences in the recognition of foreign antigen, such as those contributed by alleles at the HLA class II loci, have been proposed to affect the risk of developing cervical cancer.18, 19 Also, the risk associated with certain HLA class II alleles has been shown to be specific to individual HPV types.20 Recently, we have shown that the 2 HLA class II alleles DRB1*1501 and DQB1*0602 increase the risk of an HPV 16 infection and thereby indirectly also the risk of cervical cancer in situ.21 In our study, we address the relationships between the HLA class II haplotype DRB1*1501-DQB1*0602 and viral load of HPV 16, as well as between viral load and time span of an HPV infection.
MATERIAL AND METHODS
The participants in this retrospective case/control study were selected from a cohort of women residing in Uppsala County, Sweden, from 1969–1995.22 The participants had to fulfill the following entry criteria: (i) born in Sweden; (ii) less than 50 years of age at entry; (iii) their first smear classified as normal by cytology on squamous cell (PAP = 1). Histologic specimens (either a small biopsy or a complete “cone”) from all eligible cases were reviewed by an experienced pathologist (Jan Pontén). All cases with invasive cancer or adenocarcinoma were excluded from our study, and only cases with squamous cell cancer in situ, CIS, were included. For each case, 5 separate controls, individually matched by date of entry to the cohort (±90 days) and by year of birth, were randomly selected from the study cohort. The women included as controls had to be alive without having developed cancer in situ or invasive cancer before the date of diagnosis for their corresponding case. Also, the first smear of the cases and controls had to be classified as normal by cytology on squamous cell (PAP = 1).6, 23
Collection of Smears
The archival smears included in our study were collected during the period of 1969–1995. A total of 3,480 smears with a mean of 3.1 smears per control and 4.4 smears per case were included.
DNA was purified from Papanicolaou-stained archival smears, as previously described.22, 24
Taqman assays for detection and quantification of HPV 16 and β-actin were performed as previously described.5, 24 The β-actin estimate was used to assess differences in the amount of cells studied in individual samples. The amount of DNA in a sample is expressed as the PCR cycle number where the fluorescence surpasses the threshold (Ct). A lower Ct indicates a higher amount of DNA.
One smear, with the lowest β-actin value (highest amount of genomic DNA) based on the Taqman assay, was selected from each woman for HLA typing. Two microliters of the DNA from each smear was used for PCR amplification of the DQB1 and DRB1 loci, using biotinylated primers. The PCR products were hybridised to reverse-dotblots containing sequence-specific oligonucleotides, as provided by the manufacturer (Dynal RELI™ SSO by DYNAL, Oslo, Norway). These procedures have been described previously.25, 26
Women lacking HPV 16 data were excluded from all analyses. A t-test was used to compare the mean HPV 16 titer in carriers and noncarriers of the particular HLA haplotype studied independent of case/control status, using the SAS procedure “TTEST”. To study the relationship between HLA haplotype carrier frequency and HPV 16 titer, Mantel-Haenszel statistics were used. Mantel-Haenszel statistics were computed using the SAS procedure “FREQ”, using case/control status as confounding variable, carrier status of the haplotype or allele as row-variable and Ct-category as column-variable. The relationship between infection history and HPV titer was studied using a t-test. For calculation of positive predictive values, an incidence of cervical cancer in situ of 3% in Sweden was assumed. The predictive values were then calculated for each titer group specifically.
The cohort study was composed of 478 women diagnosed with cervical cancer in situ and 608 age-matched controls.5 HLA-DRB1 typing results were obtained for 433 cases and 469 controls and HLA-DQB1 typing results were obtained for 442 cases and 482 controls. Due to poor DNA quality, incomplete typing results (only 1 of the 2 alleles could be detected) were obtained for 6 cases and 12 controls for DRB1 and 9 cases and 17 controls for DQB1. Since the statistical analyses were based on HLA carrier frequency, these individuals were included. In total, 60% (266/441) of the cases and 16% (80/487) of the controls tested HPV 16-positive. In a previous study of this cohort,21 we concluded that the HLA class II alleles DQB1*0602 and DRB1*1501 were associated primarily with infection by HPV rather than with development of cervical cancer in situ. Therefore, for most of the following analyses of the relationships between HPV titer and HLA haplotype, we combined the cases and controls. Also, the DQB1*0602 and DRB1*1501 alleles occur frequently on the same haplotype, and their individual effect is therefore difficult to distinguish. In our study material, 93% of DRB1*1501 carriers also have the DQB1*0602 allele and also 93% of the DQB1*0602 carriers have the DRB1*1501 allele. Thus in most of the analyses, we consider the joint effect of the 2 alleles on this haplotype.
First, comparisons of the DQB1*0602-DRB1*1501 haplotype show that carriers (combining cases and controls) have a significantly higher mean HPV 16 titer as compared to noncarriers (t-test with unequal variance, p = 0.017). Also, when analysing each allele on its own, both DQB1*0602 and DRB1*1501 show significant difference in titer (t-test with unequal variance, p = 0.017 and p = 0.044, respectively). To view the relationship between the HLA genotype and HPV titer, all HPV 16-positive women were divided into 5 percentiles with respect to their viral load, where the titer was calculated as the mean Ct-value for all HPV 16-positive smears from a woman (Fig. 1, Table I). This figure shows that there is an increase in haplotype carrier frequency with higher viral load. As the smears were studied for the amount of a nuclear gene (β-actin), it was possible to look for systematic differences in cellular DNA amounts between HPV titer groups, potentially due to variation in sampling over time. The β-actin Ct values for the smears are very similar between the percentiles, and there is no indication of a systematic difference in DNA amount between the titer groups (Table I). A significant positive association was found between carrier frequency of the DQB1*0602-DRB1*1501 haplotype and the HPV 16 titer (Mantel-Haenszel statistics, controlling for case/control status, p = 0.005). Analyses of the 2 HLA alleles individually produced similar results (DQB1*0602, p = 0.006 and DRB1*1501, p = 0.001). This shows that carrying even a single copy of the DQB1*0602-DRB1*1501 haplotype is associated with a higher HPV 16 copy number. Previous studies of HPV 16 viral titer in this cohort has shown that viral load is increased in these women, even in the first smears taken from each woman.5, 6 This first smear had a normal cytology and was taken on average 8 years prior to the diagnosis of cancer in situ. A similar positive association of DRB1*1501 DQB1*0602 carrier frequency with higher HPV 16 titer is seen when we use the first positive smear from each woman (Fig. 2) (Mantel-Haenszel statistics, controlling for case/control status p = 0.004). Thus, the association between the HLA haplotype and viral titer is seen early in infection history.
Table I. Distribution of DRB1∗1501-DQB1∗0602 Haplotype Carrier Frequency and Mean β-actin Ct-values in the Different Titer Groups
*1501–*0602 carriers n (cases)
All women n (cases)
β-actin mean Ct-value
For each HPV 16-positive woman, the mean Ct-value for all HPV 16 smears was calculated. Women were then divided into different percentiles (titer groups 1–5) depending on their mean Ct-value. HPV-negative women were included in titer group 0. The β-actin estimate was used to assess differences in the amount of cells studied between titer groups.
0. Ct = 50 (HPV neg)
1. 48.47 < Ct < 50
2. 45.66 < Ct ≤ 48.47
3. 41.79 < Ct ≤ 45.66
4. 39.11 < Ct ≤ 41.79
5. Ct ≤ 39.11
To determine whether length of infection is associated with HPV titer, women were classified into one group with long-term infections (infection period more than 1 year) and a second group with short-term infections (infection period less than 1 year) (Fig. 3). Women in the group with long-term infections had at least 2 consecutive HPV 16-positive smears and a maximum of 2 years between positive smears (88 cases and 1 control). This group had higher viral load compared to women with other infection histories (t-test with unequal variance, p = 0.0001). Women in the group with short-term infections had at least 1 HPV 16-positive smear and a maximum of 2 years from the first HPV 16-positive smear to the following negative smear, with a maximum of 6 months from first to last HPV 16-positive smear (36 cases and 24 controls). This group had lower viral load compared to women with other infection histories (t-test with unequal variance, p = 0.0001).
We have shown that there is an association between HLA class II haplotype and the titer of an infecting virus, and more specifically that carriers of the haplotype DRB1*1501-DQB1*0602 have higher HPV 16 titers relative to carriers of other haplotypes. The association of the DRB1*1501-DQB1*0602 haplotype with higher viral titer was seen both using the mean HPV 16 titer for each woman and the first HPV 16-positive smear. This association was also seen when analysing each allele individually. Taken together, these results imply that the DRB1*1501-DQB1*0602 haplotype affects the HPV viral load already at stages when no cytologic changes can be detected in the epithelial cells. Our results also show that women with high HPV 16 titers are prone to long-term, and perhaps persistent or chronic, infection. Thus, the ability of an individual to control viral infection, resulting in a high or low titer, correlates with the time span of the infection. The original study design was not optimal for measuring short- or long-term infection since other studies have shown that women who clear their infection do this within 1 year. In our retrospective study, the women included attended the national screening program with sampling every third year. However, a smaller sample of women with more frequent sampling gave us the possibility to divide them into short- and long-term infection groups that roughly correlate with reported time spans for cleared or persistent infections.27, 28
The means by which the DRB1*1501-DQB1*0602 haplotype results in a higher HPV 16 titer is not known. In other studies of the HLA presentation of HPV 16 E6 peptides to the immune system, significant differences have been seen in the sensitivity of the immune system to different HLA/E6 peptide combinations.29 Also, the importance of a cytotoxic T-lymphocyte response to E6 antigens for the clearance of HPV 16 infections has been demonstrated.28 An increased risk of developing cancer for carriers of different HPV 16 E6 variants has been reported.30, 31 Also, a specific HLA haplotype (DR*04-DQ*03) in association with the HPV 16 E6 L83V variant appears to confer an increased risk of cancer development.32 Similarly, in our study, carriers of the DRB1*1501-DQB1*0602 haplotype may be compromised in their ability to present certain HPV 16 E6 antigens, rendering them more susceptible to higher HPV titer, persistent infection and thereby also more likely to develop cervical cancer. A high HPV titer and persistent infection both increases the probability of integration of HPV into the host genome and the subsequent development of cervical tumors. In HIV, different HLA profiles have similarly been described to affect viral load and disease progression, but the actual HLA alleles involved have not been pinpointed.33, 34
Our study shows that genetic factors influencing HPV titer indirectly predispose to carcinoma in situ. In view of this, the diagnostic potential of using HPV titer and host genetic susceptibility factors, such as the HLA class II alleles, for early detection and prevention of cervical cancer should be evaluated. Although the carrier frequency for the DRB1-DQB1 haplotype is quite high (25–45%), only a small number of these carriers develop carcinoma in situ, potentially resulting in a low predictive value for this marker alone. To assess the value of using DRB1*1501-DQB1*0602 carrier status together with HPV 16 titer to predict the risk of developing cervical cancer, we calculated the positive predictive value (PPV), based on the cohort in our study and using additional information for the population from which the cohort was derived. The results indicate that in the case that the HPV 16 titer has been estimated, there is no measurable added value of using DRB1*1501-DQB1*0602 carrier status in predicting the risk of developing cervical cancer (Fig. 4). Indeed, when analysing the association of case/control status and viral load with the HLA haplotype as confounding factor, HLA does not affect the outcome. This further supports our conclusion that the HLA haplotype affects the probability of an HPV 16 infection and an increased viral load and only indirectly affects the development of cancer. However, the HLA class II variants discussed are likely to account for only a portion of the genetic susceptibility to cervical cancer.35 Additional genetic host factors, involving loci affecting the immune response, such as TNFA,36 or as yet unidentified loci, may contribute to the susceptibility of cervical cancer and represent important diagnostic targets.
We thank Dr. P. Magnusson for constructive suggestions regarding the statistics and for comments on the manuscript and Dr. H. Erlich for generous support during the HLA typing.